The present invention relates to a method of testing the presence of a gene responsible for a pathological state, or the pathological state itself, in biological material in which the molecules are regularly arrayed. Specifically, the present invention relates to analysing hair for detecting the presence of BRCA I gene, the presence of breast cancer per se, the presence of prostate cancer and a confirmatory diagnosis for Alzheimer""s disease.
Breast cancer is still a major cause of death in women in Australia. It has been established that approximately 14% of Australian women will have it during their life-time. However, if the cancer is diagnosed early enough, while it is still contained within the breast, there is approximately 98% chance of obtaining a total cure. An early low-cost diagnostic tool would be a major break-through in this area of medicine.
The genes responsible for familial breast cancer have already been located and clinics are being formed to closely monitor those whose family members have been shown to test positive to the gene. These tests however take weeks to complete, are extremely costly and cannot therefore be extended to all women and are at present limited to the high risk group of persons whose mothers had breast cancer.
At present a very expensive and time consuming test is used to determine the carriers of genes, such as breast cancer genes.
The technique of this invention provides a relatively low cost, approximately 1 minute alternative test. It also offers a possible mass screening technique for example, for breast cancer per se with easy access to sample collection and handling. Such a test may replace mammography which is not only expensive, requires attendance at various locations and from the woman""s point of view is quite painful but also is not 100% reliable and in some cases very difficult to interpret.
Considered at the microscopic level, a single hair consists of a core (the medulla) surrounded by an intermediate layer (the cortex) and then by a thin outer envelope (the cuticle). The cortex can be considered as a continuous network of xcex1-keratin fibrils arranged parallel to the length of the hair and held together by a cell membrane complex. During keratinisation in the follicle each individual cortical cell is wrapped in a plasma membrane, which is basically a biomolecular layer of phospholipids, which acts as the adhesive between neighbouring cells. Such a regular array is ideally suited to fibre x-ray diffraction analysis, and the ultrastructure of human hair has been previously determined from high-intensity synchrotron radiation scattering data combined with results from physical swelling and stretching experiments. These data show the major components of the cortex to be hexagonal arrays of xcex1-keratin fibrils comprising the intermediate filaments (IFs). The fundamental unit of the IF is the double helical xcex1-keratin chain. Two of these coils together to form a tightly bound tetramer. Eight of the tetramers then wind in a slow helix to form the outer layer of the IF. The eight tetrameric units are staggered so that the commencement of the seventh is directly above the first and the whole assembly is tilted at an angle of 7xc2x0, thus providing the six linkages in one complete turn around the IF to form an hexagonal array. The xcex1-helical units of the IFs with their shorter linker unit consist of around 311-312 residues, which corresponds to the 47.0 nm repeat in the fibre direction. The axial repeat of the linkage attachment points, at 62.7 nm in the fibre direction, correspond to the non-helical tail repeats along the inclined tetrameric units. A typical x-ray diffraction pattern obtained from normal subjects between the ages of 4 and 80 is given in FIG. 2, that from persons with breast cancer is given in FIG. 3.
Low angle fibre X-ray diffraction along with appropriate analysis will yield the molecular structure of any material in which there is a regular array of molecules. Hair is such a material, characterised by regular arrays of xcex1-keratin. Studies using synchrotron radiation have yielded not only the structure of xcex1-keratin per se and that of the specific xcex1-keratin in hair but have also revealed and identified the molecular changes that occur in human hair in insulin dependent diabetes.
It has now been surprisingly found that fibre x-ray diffraction studies using synchrotron radiation can reveal clear and consistent changes in the ultrastructure of hair from breast cancer patients.
According to a first aspect of the present invention, there is provided a method for detecting the presence of a gene responsible for a pathological state; or the pathological state itself in a patient comprising exposing at least one hair from the patient to fibre x-ray diffraction and detecting changes in the ultrastructure of the hair.
According to a second aspect of the present invention, there is provided an instrument for detecting the presence of a gene responsible for a pathological state; or the pathological state itself, using a hair sample, comprising: an X-ray source producing a beam of X-ray radiation; a sample stage for positioning said hair sample within said beam; a detector to detect the scattering of said X-ray beam caused by said hair sample; and a display means associated with said detector for displaying the output thereof; whereby patterns of output related to the presence of said gene or said pathological state are displayed for interpretation.
Suitably, the X-ray used are derived from synchrotron radiation or other monochromatic X-ray sources providing X-rays within the energy range of five to twenty-five keV.
In practice, a single hair is taken from adjacent to the scalp or other areas such as the pubic area of a person. The sample is washed in distilled water and dried under normal conditions (temperature 20xc2x0 C. and atmospheric pressure), and then cut into approximately 30 mm lengths. The specimen is mounted in a cell, under sufficient tension to maintain alignment. The cell is then placed on a low-angle diffractometer so that the sample is normal to the beam. The space between the sample and the imaging plate is evacuated so as to minimise absorption losses and air scattering.
The X-ray diffraction experiments are carried out using a monochromatic X-ray source such as a low-angle synchrotron facility, for example at B115, Photon Factory, Tsukuba or the like with an X-ray wavelength ranging between 0.06 and 0.20 nm. An incident flux at the specimen of approximately 8xc3x971010 photons/sec can be generated when the Photon Factory storage ring is operated at 25 GeV with a beam current of 145 mA. A 0.15 nm wave-length was used at this facility. The X-ray patterns are recorded on imaging plates, for example Fuji Bas III imaging plates. Exposure time for the hair sample is suitably between 5 seconds and 5 minutes using synchrotron sources, but may be days or weeks using other X-ray sources. Preferably, the exposure time is approximately 60 seconds on second generation synchrotron sources and 20 seconds on third generation synchrotron sources. Sample to imaging plate distances are approximately 20 mm to 3000 mm. Suitably 400 mm is used. The analysis of the recorded patterns is carried out using two computer packages. The meridional data is analysed using a Bragg analysis, the equatorial data is analysed using appropriate Bessel functions.